1. Field of the Invention
The present invention relates to devices and methods in horizontal submarine gel electrophoresis. In particular, the present invention relates to devices and methods for decreasing temperature in gel matrix so that higher voltage can be applied to the gel matrix in horizontal submarine gel electrophoresis.
2. Background of the Invention
Gel electrophoresis is one of the most commonly utilized tools in biomedical researches and industries. In gel electrophoresis, a sample mixture of biomolecules is applied to a gel matrix and an electric field is also applied to the gel matrix via a buffer system. The charged components in the sample mixture migrate through the gel matrix at different migration rates so that they can be separated from each other after electrophoresis. The migration rate of the charged components is directly related to the voltage of the electric field applied. Higher voltage generates higher migration rate under a given condition.
Gel matrixes used in electrophoresis are made from their gel solutions via different mechanisms. Polyacrylamid gel matrix is formed from a gel solution by a chemical reaction. Agarose gel matrix, in contrast, is formed from a gel solution by a decrease in temperature. The from agarose gel matrix is readily reversible whenever the temperature rises to a certain point. The properties of the agarose gel matrix are significantly altered by a temperature elevation due to the nature of the gel forming mechanism. Thus, it is critical for agarose gel electrophoresis to maintain the gel matrix at a low temperature.
Conventional horizontal submarine gel electrophoresis is the most commonly used format for agarose gel due to its simplicity in gel formation and manipulation. The gel matrix is usually casted in an open tray and the tray with the gel is then immersed into a buffer system for applying an electric field to the gel matrix and absorbing heat from the gel matrix. A typical feature of this format is that a heavy electric current passes through two pathways, a gel pathway through the gel matrix and a buffer pathway through the buffer around the gel matrix. The heavy electric current causes a massive heat generation during electrophoresis.
Gel pathway and buffer pathway are two partners in forming electric circuit. The condition of the buffer pathway around the gel matrix affects the result of the electrophoresis in the gel matrix due to their partnership relation. Thus, the consistency of electrophoresis can be ruined by buffer related manual variations. Such as the volume of the buffer used, the level of the buffer surface adjusted, and the relative ionic strength between the buffer and the gel matrix.
The heavy electric current causes the massive heat generation during electrophoresis. The massive heat must be removed from the gel matrix in order to reduce the temperature of the gel matrix. But, unfortunately, a logic paradox makes the temperature reduction a difficulty in conventional horizontal submarine gel electrophoresis, that is, the temperature of the gel matrix will be further reduced if more buffer is used to cover the gel matrix and the temperature of the gel matrix will be further elevated if more buffer is used to cover the gel matrix. It is a fact that more buffer can absorb more heat from the gel matrix. It is also a fact that more butter will lead to a heavier electric current.
The existence of the buffer pathway around the gel matrix results in disadvantages:
(1) The electrophoresis has to be a slow process because the voltage applied must be limited to a low scale level to avoid generating excess heat which will otherwise distort the gel matrix. PA1 (2) A strong buffering capacity is required for the buffer system to maintain a pH balance under the heavy electric current. PA1 (3) A careful manipulation of adjusting buffer condition is required for reducing the variation of electrophoresis.
These disadvantages are long-problems. Attempts have been made for pursuing improvement of the conventional horizontal submarine gel electrophoresis.
Audeh. U.S. Pat. No. 4,702,814, teaches a horizontal submarine gel device having a gas collecting means and a conduit for eliminating the requirement of having a strong buffering capacity in buffer system. But Audeh fails to recognize the basis of those disadvantages. A buffer pathway is inherited in his device. Thus, Audeh fails to accelerate the slow process of electrophoresis and fails to omit the requirement of adjusting the buffer condition.
Hoefer, 1994 Catalog of Hoefer Scientific Instruments at page 30, teaches a horizontal submarine gel device having a coolant mixture in base chamber for accelerating the slow process of electrophoresis. Hoefer, however, also fails to recognize the basis of those disadvantages. The buffer pathway is still inherited in his device. Hence, Hoefer fail to omit the requirement of adjusting the buffer condition and fails to eliminate the requirement of having strong buffering capacity for maintaining pH balance. Furthermore, Hoefer fails to optimize the acceleration of the slow process of electrophoresis. First, a massive heat is still generating from the buffer pathway around the gel matrix. Secondly, the heat absorption is dramatically barricaded by placing a plastic layer of gel tray and a plastic layer of base chamber between the gel matrix and the coolant. The acceleration is therefore a limited improvement.
Fairfield, U.S. Pat. No. 5,074,981, teaches a device and method for high speed gel electrophoresis. Fairfield has recognized the basis of those disadvantages so that the buffer pathway is removed from his device and method. Bin, Fairfield fails to find a correct solution for most routine applications. To reach his high speed, Fairfield sacrifices the most attractive advantage of conventional horizontal submarine gel electrophoresis, the simplicity, and replaces it with a series of delicate requirements. Such as a series of additional devices, a series of difficulties in manipulation, a series of time-consuming steps, and a series of risks of failure. For example, Fairfield requires a vacuum grease, an external pump, an ice bag, and a thermometer. Fairfield has to deal with difficulties of handling a thin agarose gel, of assembling ice bag over the thin agarose gel without disturbing applied sample, and of controlling all samples into a volume less than 4 .mu.l. Fairfield needs time to prepare all his samples to high concentration in order to load enough amount of sample in a volume less than 4 .mu.l, to cover the gel tightly with plastic wrap, to place gel in 4.degree. C. for at least 30 minutes, and to assemble all required devices together carefully. Fairfield may easily fail by a series of uncertainties, such as the failure in ice crystals penetrating into the gel at -20.degree. C., the failure in making an essential excellent thermal contact at all layers, and the failure in disturbing applied sample during layer assembly.
Fairfield accelerates the slow process of electrophoresis by paying a tremendous price. Such overweight price is constructed as a limiting barrier of its acceptability for most routine applications. Besides, the acceleration does not save time for the whole procedure because Fairfield has to spend a long time to perform his device and method. Fairfield also fails to improve the consistency of electrophoresis because much more manual uncertainties have been introduced in his device and method.
A horizontal submarine gel electrophoresis device and method with its original simplicity but without those disadvantages is highly desirable but the long-felt problem remains unsolved.
It is, thereafter, an object of the present invention to remove the basis of those disadvantages, the buffer pathway, while maintaining its original simplicity so that a simple device and method for most routine applications can be provided.